Glycine-induced long-term potentiation is associated with structural and functional modifications of α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid receptors

Global long-term potentiation (LTP) was induced in organotypic hippocampal slice cultures by a brief application of 10 mM glycine. Glycine-induced LTP was occluded by previous theta burst stimulation-induced potentiation, indicating that both phenomena share similar cellular processes. Glycine-induced LTP was associated with increased [3H]α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA) binding in membrane fractions as well as increased amount of a selective spectrin breakdown product generated by calpain-mediated spectrin proteolysis. Antibodies against the C-terminal (C-Ab) and N-terminal (N-Ab) domains of GluR1 subunits were used to evaluate structural changes in AMPA receptor properties resulting from glycine-induced LTP. No quantitative or qualitative changes were observed in Western blots from membrane fractions prepared from glycine-treated slices with C-Ab. In contrast, Western blots stained with N-Ab revealed the formation of a 98-kDa species of GluR1 subunits as well as an increased amount of immunoreactivity after glycine-induced LTP. The amount of spectrin breakdown product was positively correlated with the amount of the 98-kDa species of GluR1 after glycine treatment. Functional modifications of AMPA receptors were evaluated by determining changes in the effect of pressure-applied AMPA on synaptic responses before and after glycine-induced LTP. Glycine treatment produced a significant increase in AMPA receptor function after potentiation that correlated with the degree of potentiation. The results indicate that LTP induction produces calpain activation, truncation of the C-Ab domain of GluR1 subunits of AMPA receptors, and increased AMPA receptor function. They also suggest that insertion of new receptors takes place after LTP induction.

Evaluating treatment protocols to prevent antibiotic resistance

The spread of bacteria resistant to antimicrobial agents calls for population-wide treatment strategies to delay or reverse the trend toward antibiotic resistance. Here we propose new criteria for the evaluation of the population-wide effects of treatment protocols for directly transmitted bacterial infections and discuss different usage patterns for single and multiple antibiotic therapy. A mathematical model suggests that the long-term benefit of single drug treatment from introduction of the antibiotic until a high frequency of resistance precludes its use is almost independent of the pattern of antibiotic use. When more than one antibiotic is employed, sequential use of different antibiotics in the population (“cycling”) is always inferior to treatment strategies where, at any given time, equal fractions of the population receive different antibiotics. However, treatment of all patients with a combination of antibiotics is in most cases the optimal treatment strategy.

Long-term exposure to high corticosterone levels attenuates serotonin responses in rat hippocampal CA1 neurons

Recent studies indicated that hyperactivity of the hypothalamo-pituitary-adrenal system is a considerable risk factor for the precipitation of affective disorders, most notably of major depression. The mechanism by which this hyperactivity eventually leads to clinical symptoms of depression is unknown. In the present animal study, we tested one possible mechanism, i.e., that long-term exposure to high corticosterone levels alters functional responses to serotonin in the hippocampus, an important area in the etiology of depression. Rats were injected daily for 3 weeks with a high dose of corticosterone; electrophysiological responses to serotonin were recorded intracellularly from CA1 pyramidal neurons in vitro. We observed that daily injections with corticosterone gradually attenuate the membrane hyperpolarization and resistance decrease mediated by serotonin-1A receptors. We next used single-cell antisense RNA amplification from identified CA1 pyramidal neurons to resolve whether the functional deficits in serotonin responsiveness are accompanied by decreased expression levels of the serotonin-1A receptor. It appeared that expression of serotonin-1A receptors in CA1 pyramidal cells is not altered; this result was supported by in situ hybridization. Expression of corticosteroid receptors in the same cells, particularly of the high-affinity mineralocorticoid receptor, was significantly reduced after long-term corticosterone treatment. The present findings indicate that prolonged elevation of the corticosteroid concentration, a possible causal factor for major depression in humans, gradually attenuates responsiveness to serotonin without necessarily decreasing serotonin-1A receptor mRNA levels in pyramidal neurons. These functional changes may occur by a posttranscriptional mechanism or by transcriptional regulation of genes other than the serotonin-1A receptor gene itself.

Specific therapy regimes could lead to long-term immunological control of HIV

We use mathematical models to study the relationship between HIV and the immune system during the natural course of infection and in the context of different antiviral treatment regimes. The models suggest that an efficient cytotoxic T lymphocyte (CTL) memory response is required to control the virus. We define CTL memory as long-term persistence of CTL precursors in the absence of antigen. Infection and depletion of CD4+ T helper cells interfere with CTL memory generation, resulting in persistent viral replication and disease progression. We find that antiviral drug therapy during primary infection can enable the development of CTL memory. In chronically infected patients, specific treatment schedules, either including deliberate drug holidays or antigenic boosts of the immune system, can lead to a re-establishment of CTL memory. Whether such treatment regimes would lead to long-term immunologic control deserves investigation under carefully controlled conditions.

Rolipram, a type IV-specific phosphodiesterase inhibitor, facilitates the establishment of long-lasting long-term potentiation and improves memory

In an attempt to improve behavioral memory, we devised a strategy to amplify the signal-to-noise ratio of the cAMP pathway, which plays a central role in hippocampal synaptic plasticity and behavioral memory. Multiple high-frequency trains of electrical stimulation induce long-lasting long-term potentiation, a form of synaptic strengthening in hippocampus that is greater in both magnitude and persistence than the short-lasting long-term potentiation generated by a single tetanic train. Studies using pharmacological inhibitors and genetic manipulations have shown that this difference in response depends on the activity of cAMP-dependent protein kinase A. Genetic studies have also indicated that protein kinase A and one of its target transcription factors, cAMP response element binding protein, are important in memory in vivo. These findings suggested that amplification of signals through the cAMP pathway might lower the threshold for generating long-lasting long-term potentiation and increase behavioral memory. We therefore examined the biochemical, physiological, and behavioral effects in mice of partial inhibition of a hippocampal cAMP phosphodiesterase. Concentrations of a type IV-specific phosphodiesterase inhibitor, rolipram, which had no significant effect on basal cAMP concentration, increased the cAMP response of hippocampal slices to stimulation with forskolin and induced persistent long-term potentiation in CA1 after a single tetanic train. In both young and aged mice, rolipram treatment before training increased long- but not short-term retention in freezing to context, a hippocampus-dependent memory task.

Long-term correction of obesity and diabetes in genetically obese mice by a single intramuscular injection of recombinant adeno-associated virus encoding mouse leptin

The ob/ob mouse is genetically deficient in leptin and exhibits a phenotype that includes obesity and non-insulin-dependent diabetes melitus. This phenotype closely resembles the morbid obesity seen in humans. In this study, we demonstrate that a single intramuscular injection of a recombinant adeno-associated virus (AAV) vector encoding mouse leptin (rAAV-leptin) in ob/ob mice leads to prevention of obesity and diabetes. The treated animals show normalization of metabolic abnormalities including hyperglycemia, insulin resistance, impaired glucose tolerance, and lethargy. The effects of a single injection have lasted through the 6-month course of the study. At all time points measured the circulating levels of leptin in the serum were similar to age-matched control C57 mice. These results demonstrate that maintenance of normal levels of leptin (2–5 ng/ml) in the circulation can prevent both the onset of obesity and associated non-insulin-dependent diabetes. Thus a single injection of a rAAV vector expressing a therapeutic gene can lead to complete and long-term correction of a genetic disorder. Our study demonstrates the long-term correction of a disease caused by a genetic defect and proves the feasibility of using rAAV-based vectors for the treatment of chronic disorders like obesity.

Adeno-associated viral vector-mediated gene transfer results in long-term enzymatic and functional correction in multiple organs of Fabry mice

Fabry disease is a lysosomal storage disorder caused by a deficiency of the lysosomal enzyme α-galactosidase A (α-gal A). This enzyme deficiency leads to impaired catabolism of α-galactosyl-terminal lipids such as globotriaosylceramide (Gb3). Patients develop painful neuropathy and vascular occlusions that progressively lead to cardiovascular, cerebrovascular, and renal dysfunction and early death. Although enzyme replacement therapy and bone marrow transplantation have shown promise in the murine analog of Fabry disease, gene therapy holds a strong potential for treating this disease in humans. Delivery of the normal α-gal A gene (cDNA) into a depot organ such as liver may be sufficient to elicit corrective circulating levels of the deficient enzyme. To investigate this possibility, a recombinant adeno-associated viral vector encoding human α-gal A (rAAV-AGA) was constructed and injected into the hepatic portal vein of Fabry mice. Two weeks postinjection, α-gal A activity in the livers of rAAV-AGA-injected Fabry mice was 20–35% of that of the normal mice. The transduced animals continued to show higher α-gal A levels in liver and other tissues compared with the untouched Fabry controls as long as 6 months after treatment. In parallel to the elevated enzyme levels, we see significant reductions in Gb3 levels to near normal at 2 and 5 weeks posttreatment. The lower Gb3 levels continued in liver, spleen, and heart, up to 25 weeks with no significant immune response to the virus or α-gal A. Also, no signs of liver toxicity occurred after the rAAV-AGA administration. These findings suggest that an AAV-mediated gene transfer may be useful for the treatment of Fabry disease and possibly other metabolic disorders.

Cloning and characterization of a hormonally regulated rat long chain acyl-CoA synthetase

A previously unidentified gonadotropin-regulated long chain acyl-CoA synthetase (GR-LACS) was cloned and characterized as a 79-kDa cytoplasmic protein expressed in Leydig cells of the rat testis. GR-LACS shares sequence identity with two conserved regions of the LACS and luciferase families, including the ATP/AMP binding domain and the 25-aa fatty acyl-CoA synthetase signature motif, but displays low overall amino acid similarities (23–28%). GR-LACS mRNA is expressed abundantly in Leydig cells of the adult testis and to a lesser degree in the seminiferous tubules in spermatogonia and Sertoli cells. It is also observed in ovary and brain. Immunoreactive protein expression was observed mainly in Leydig cells and minimally in the tubules but was not detected in other tissues. In vivo, treatment with a desensitizing dose of human chorionic gonadotropin caused transcriptional down-regulation of GR-LACS expression in Leydig cells. The expressed protein present in the cytoplasm of transfected cells displayed acyl-CoA synthetase activity for long chain fatty acid substrates. GR-LACS may contribute to the provision of energy requirements and to the biosynthesis of steroid precursors and could participate through acyl-CoA's multiple functions in the regulation of the male gonad.

(+)-Abscisic Acid Metabolism, 3-Ketoacyl-Coenzyme A Synthase Gene Expression, and Very-Long-Chain Monounsaturated Fatty Acid Biosynthesis in Brassica napus Embryos1

Microspore-derived embryos of Brassica napus cv Reston were used to examine the effects of exogenous (+)-abscisic acid (ABA) and related compounds on the accumulation of very-long-chain monounsaturated fatty acids (VLCMFAs), VLCMFA elongase complex activity, and induction of the 3-ketoacyl-coenzyme A synthase (KCS) gene encoding the condensing enzyme of the VLCMFA elongation system. Of the concentrations tested, (+)-ABA at 10 μm showed the strongest effect. Maximum activity of the elongase complex, observed 6 h after 10 μm (+)-ABA treatment, was 60% higher than that of the untreated embryos at 24 h. The transcript of the KCS gene was induced by 10 μm (+)-ABA within 1 h and further increased up to 6 h. The VLCMFAs eicosenoic acid (20:1) and erucoic acid (22:1) increased by 1.5- to 2-fold in embryos treated with (+)-ABA for 72 h. Also, (+)-8′-methylene ABA, which is metabolized more slowly than ABA, had a stronger ABA-like effect on the KCS gene transcription, elongase complex activity (28% higher), and level of VLCMFAs (25–30% higher) than ABA. After 24 h approximately 60% of the added (+)-[3H]ABA (10 μm) was metabolized, yielding labeled phaseic and dihydrophaseic acid. This study demonstrates that (+)-ABA promotes VLCMFA biosynthesis via increased expression of the KCS gene and that reducing ABA catabolism would increase VLCMFAs in microspore-derived embryos.

Effects of Short- and Long-Term Elevated CO2 on the Expression of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Genes and Carbohydrate Accumulation in Leaves of Arabidopsis thaliana (L.) Heynh.1

To investigate the proposed molecular characteristics of sugar-mediated repression of photosynthetic genes during plant acclimation to elevated CO2, we examined the relationship between the accumulation and metabolism of nonstructural carbohydrates and changes in ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) gene expression in leaves of Arabidopsis thaliana exposed to elevated CO2. Long-term growth of Arabidopsis at high CO2 (1000 μL L−1) resulted in a 2-fold increase in nonstructural carbohydrates, a large decrease in the expression of Rubisco protein and in the transcript of rbcL, the gene encoding the large subunit of Rubisco (approximately 35–40%), and an even greater decline in mRNA of rbcS, the gene encoding the small subunit (approximately 60%). This differential response of protein and mRNAs suggests that transcriptional/posttranscriptional processes and protein turnover may determine the final amount of leaf Rubisco protein at high CO2. Analysis of mRNA levels of individual rbcS genes indicated that reduction in total rbcS transcripts was caused by decreased expression of all four rbcS genes. Short-term transfer of Arabidopsis plants grown at ambient CO2 to high CO2 resulted in a decrease in total rbcS mRNA by d 6, whereas Rubisco content and rbcL mRNA decreased by d 9. Transfer to high CO2 reduced the maximum expression level of the primary rbcS genes (1A and, particularly, 3B) by limiting their normal pattern of accumulation through the night period. The decreased nighttime levels of rbcS mRNA were associated with a nocturnal increase in leaf hexoses. We suggest that prolonged nighttime hexose metabolism resulting from exposure to elevated CO2 affects rbcS transcript accumulation and, ultimately, the level of Rubisco protein.

The application of genetically engineered herpes simplex viruses to the treatment of experimental brain tumors.

Due to lack of effective therapy, primary brain tumors are the focus of intense investigation of novel experimental approaches that use vectors and recombinant viruses. Therapeutic approaches have been both indirect, whereby vectors are used, or direct to allow for direct cell killing by the introduced virus. Genetically engineered herpes simplex viruses are currently being evaluated as an experimental approach to eradicate malignant human gliomas. Initial studies with gamma (1)34.5 mutants, R3616 (from which both copies of the gamma (1)34.5 gene have been deleted) and R4009 (a construct with two stop codons inserted into the gamma (1)34.5 gene), have been assessed. In a syngeneic scid mouse intracranial tumor model, recombinant herpes simplex virus can be experimentally used for the treatment of brain tumors. These viruses and additional engineered viruses were subsequently tested in human glioma cells both in vitro and in vivo. Using a xenogeneic scid mouse intracranial glioma model, R4009 therapy of established tumors significantly prolonged survival. Most importantly, long-term survival was achieved, with histologic evidence that R4009 eradicated intracranial tumors in this model. Furthermore, the opportunity to evaluate gamma (1)34.5 mutants that have enhanced oncolytic activity, e.g., R8309 where the carboxyl terminus of the gamma (1)34.5 gene has been replaced by the murine homologue, MyD116, are considered.

Long-term expression of erythropoietin in the systemic circulation of mice after intramuscular injection of a plasmid DNA vector.

Erythropoietin (Epo)-responsive anemia is a common and debilitating complication of chronic renal failure and human immunodeficiency virus infection. Current therapy for this condition involves repeated intravenous or subcutaneous injections of recombinant Epo. In this report, we describe the development of a novel muscle-based gene transfer approach that produces long-term expression of physiologically significant levels of Epo in the systemic circulation of mice. We have constructed a plasmid expression vector, pVRmEpo, that contains the murine Epo cDNA under the transcriptional control of the cytomegalovirus immediate early (CMV-IE) promoter, the CMV-IE 5' untranslated region, and intron A. A single intramuscular (i.m.) injection of as little as 10 micrograms of this plasmid into immunocompetent adult mice produced physiologically significant elevations in serum Epo levels and increased hematocrits from preinjection levels of 48 +/- 0.4% to levels of 64 +/- 3.3% 45 days after injection. Hematocrits in these animals remained elevated at greater than 60% for at least 90 days after a single i.m. injection of 10 micrograms of pVRmEpo. We observed a dose-response relationship between the amount of plasmid DNA injected and subsequent elevations in hematocrits. Mice injected once with 300 micrograms of pVRmEpo displayed 5-fold increased serum Epo levels and elevated hematocrits of 79 +/- 3.3% at 45 days after injection. The i.m. injected plasmid DNA remained localized to the site of injection as assayed by the PCR. We conclude that i.m. injection of plasmid DNA represents a viable nonviral gene transfer method for the treatment of acquired and inherited serum protein deficiencies.

Long-term in vitro correction of alpha-L-iduronidase deficiency (Hurler syndrome) in human bone marrow.

Allogeneic bone marrow transplantation is the most effective treatment for Hurler syndrome but, since this therapy is not available to all patients, we have considered an alternative approach based on transfer and expression of the normal gene in autologous bone marrow. A retroviral vector carrying the full-length cDNA for alpha-L-iduronidase has been constructed and used to transduce bone marrow from patients with this disorder. Various gene-transfer protocols have been assessed including the effect of intensive schedules of exposure of bone marrow to viral supernatant and the influence of growth factors. With these protocols, we have demonstrated successful gene transfer into primitive CD34+ cells and subsequent enzyme expression in their maturing progeny. Also, by using long-term bone marrow cultures, we have demonstrated high levels of enzyme expression sustained for several months. The efficiency of gene transfer has been assessed by PCR analysis of hemopoietic colonies as 25-56%. No advantage has been demonstrated for the addition of growth factors or intensive viral exposure schedules. The enzyme is secreted into the medium and functional localization has been demonstrated by reversal of the phenotypic effects of lysosomal storage in macrophages. This work suggests that retroviral gene transfer into human bone marrow may offer the prospect for gene therapy of Hurler syndrome in young patients without a matched sibling donor.

Effects of TH1 and TH2 cytokines on CD8+ cell response against human immunodeficiency virus: implications for long-term survival.

CD8+ cells from long-term survivors [LTS; infected with human immunodeficiency virus (HIV) for 10 or more years and having CD4+ cell counts of > or = 500 cells per microliters] have a 3-fold greater ability to suppress HIV replication than do CD8+ cells from patients who have progressed to disease (progressors) during the same time period. A change in the pattern of cytokines produced in the host from those that typically favor cell-mediated immunity (T helper 1, TH1 or type 1) to those that down-regulate it (T helper 2, TH2 or type 2) was investigated as a cause of this reduced CD8+ cell anti-HIV function. Treatment of CD8+ cells from LTS with the TH1 cytokine interleukin (IL)-2 enhanced their anti-HIV activity, whereas exposure of these cells to TH2 cytokines IL-4 or IL-10 reduced their ability to suppress HIV replication and to produce IL-2. IL-2 could prevent and reverse the inhibitory effects of IL-4 and IL-10. Moreover, prolonged exposure of CD8+ cells from some progressors to IL-2 improved the ability of these cells to suppress HIV replication. These observations support previous findings suggesting that strong CD8+ cell responses play an important role in maintaining an asymptomatic state in HIV infection. The data suggest that the loss of CD8+ cell suppression of HIV replication associated with disease progression results from a shift in cytokine production within the infected host from a TH1 to a TH2 pattern. Modulation of these cytokines could provide benefit to HIV-infected individuals by improving their CD8+ cell anti-HIV activity.

Pituitary follicle-stimulating hormone (FSH) induces CREM gene expression in Sertoli cells: involvement in long-term desensitization of the FSH receptor.

Transcription factor CREM (cAMP-responsive element modulator) plays a pivotal role in the nuclear response to cAMP in neuroendocrine cells. We have previously shown that follicle-stimulating hormone (FSH) directs CREM expression in male germ cells. The physiological importance of FSH in Sertoli cell function prompted us to analyze its effect on CREM expression in these cells. We observed a dramatic and specific increase in the CREM isoform ICER (inducible cAMP early repressor) expression, with a peak 4 h after FSH treatment of primary Sertoli cells. Interestingly, induced levels of ICER protein persist for a considerably longer time. Induction of the repressor ICER accompanies early down-regulation of the FSH receptor transcript, which leads to long-term desensitization. Here we show that ICER represses FSH receptor expression by binding to a CRE-like sequence in the regulatory region of the gene. Our results confirm the crucial role played by CREM in hormonal control and suggest its role in the long-term desensitization phenomenon of peptide membrane receptors.